Asiandu et al, 2019 |Abstract 25
BIOVALENTIA: BIOLOGICAL RESEARCH JOURNAL
e-ISSN: 2477-1392
Vol.5 No.2, November 2019
Exploration of Endophytic Fungi of Dragon Scale’s Fern (Pyrrosia
Piloselloides L. M.G. Price) as an Antibacterial Sources
Angga Puja Asiandu1,Hary Widjajanti1*, Elisa Nurnawati1
Biology Department, Faculty of Mathematics and Natural Science, Sriwijaya University
*Coresponding author
E-mail address: [email protected] (Hary Widjajanti).
Peer review under responsibility of Biology Department Sriwijaya University
Abstract
Endophytic fungi are fungi which live inside the host plant tissue and have been undergone a horizontal gene transfer process.
Endophytic fungi are able to synthesize the same bioactive compounds which synthesized by their host plants. The host plant used in this research was dragon’s scales fern (Pyrrosia piloselloides (L.) M.G. Price). Dragon’s scales fern produces various of bioactive compounds which used as antibacterial agents such as polyphenols. This research was aimed to obtain endophytic fungi isolates from trophophyll fronds and sporophyll fronds of dragon’s scales fern, to determine the antibacterial activity of the secondary metabolite extracts of endophytic fungi, to determine the Minimum Inhibitory Concentration (MIC), to determine the characteristics of the endophytic fungi isolates which potentially as antibacterial source. Based on the research, 13 endophytic fungi isolates were obtained from dragon’s scales fern fronds consist of 5 isolates from trophophyll fronds and 8 isolates from sporophyll fronds. The antibacterial activity test showed that the extract of secondary metabolites of the isolate DTP2 had the highest inhibition zone diameter against E.coli 14.82 ± 4.05 mm, DTP4 against S.aureus 8.80 ± 0.03 mm and DSP4 against S.dysentriae 10.15 ± 0.36 mm.
MIC of ethyl acetate extracts of secondary metabolites of isolate DTP2 against E.coli was 125 µg/mL, DTP4 against S.aureus was 125 µg/mL and DSP4 against S.dysentriae was 31.25 µg/mL. The endophytic fungi isolate DTP2 identified as Aureobasidium melanogenum, DTP4 identified as Penicillium allii-sativi and DSP4 identified as Aspergillus flocculosus.
Keywords: Dragon’s Scales Fern, Endophytic Fungi, Secondary Metabolites, Antibacterials, Minimum Inhibitory Concentration.
Received: 21 July 2019, Accepted: 12 October 2019.
1. INTRODUCTION
Endophytic fungi are a group of fungi that spend their life cycle by colonizing the living tissue of host plants without causing a negative impact on their hosts.
Endophytic fungi have been co-evolved with their host plant and have undergone a Horizontal Gene Transfer process (Deepika et al., 2016). Endophytic fungi have a mutualism symbiotic with its host plants. Endophytic fungi are able to produce various kinds of bioactive compounds that can be used to increase host resistance to pathogenic attacks (Akmalasari et al., 2013).
The use of endophytic fungi to produce various antibacterial compounds will reduce the exploitation of host plants. It can thus preserve the host plant from the threat of extinction. The secondary metabolites produced by endophytic fungi are the right choice to overcome the resistance of pathogenic bacteria (Murdiyah, 2017).
One of the host plant that can be used as a source of endophytic fungi isolates is dragon’s scales fern (P.piloselloides (L.) M.G. Price). Dragon’s scales fern is a fern of Polypodiaceae family. This fern has been used by the community as traditional medicines for diarrhea and vaginal discharge (Oktavia et al., 2017). Dragon’s scales fronds contain various kinds of secondary metabolites which potentially as antibacterial such as flavonoids, polyphenols, tannins, monoterpenoids, saponins, triterpenoids and steroids (Saptarini et al., 2018).
This research was aimed to obtain endophytic fungi isolates from trophophyll and sporophyll fronds of dragon’s scales fern, to determine the antibacterial activity of secondary metabolites extracts of of the endophytic fungi, to determine the Minimum Inhibitory Concentration (MIC), and to determine the characteristics of endophytic fungi which are potentially as an antibacterial sources.
Asiandu et al, 2019 |Abstract 26
2. Materials And Method
This research conducted in August 2018 until January 2019 at the Microbiology Laboratory, Laboratory of Genetics and Biotechnology, Department of Biology, Faculty of Mathematics and Natural Science, Sriwijaya University. Dragon’s scale fern fronds were collected from tea plantations on mounth Dempo, Pagaralam City, South Sumatra Province. The collected dragon’s scale fern fronds were young, green and uniform size (Desiyana et al., 2016). The bacteria used in this research were Escherichia coli, Staphylococcus aureus and Shigella dysentriae.
2.1 Tools and Materials Sterilization
The media includes Czapek Dox Agar (CDA), Czapek Yeast Agar (CYA), Malt Extract Agar (MEA), Mueller Hinton Agar (MHA), Nutrient Agar (NA), Potato Dextrose Agar (PDB), and Potato Dextrose Broth (PDB) sterilized along with the glassware using an autoclave at a pressure of 15 lbs at 121 oC for 15 minutes
2.2 Sample Preparation
Dragon’s scales fronds washed using water for 5 minutes, soaked in 70% alcohol solution for 3 minutes and 5% NaClO solution for 5 minutes. The fronds are soaked back in 70% alcohol for 30 seconds, rinsed with sterile distilled water 2 times and dried on sterile tissue. The samples of dragon’s scales fronds were cut 1 cm length and crushed using mortar (Ramadhani et al., 2017).
2.3 Isolation of Endophytic Fungi
The pieces and extract of dragon’s scales fronds were placed on the surface of the PDA medium, incubated for 3-7 days at room temperature (Pratiwi et al., 2014).
Fungal colonies that have the same shape, color and size are considered as the same isolates. Every colony which has different characteristics were purified (Suciatmih et al., 2011).
2.4 Purification and Stock Preparation of Endophytic Fungi
The mycelium of Endophytic fungi that grew in the surrounding area of fronds samples in the media was taken using sterile osseous needles and transferred aseptically to new petri dishes filled with sterile PDA, incubated at room temperature for 48-72 hours.
Endophytic fungi stocks were made by inoculating pure endophytic fungi isolates into the oblique PDA medium (Widowati et al., 2016).
2.5 Cultivation and Extraction of Secondary Metabolites of Endophytic Fungi
7 days old pure endophytic fungi culture was taken 10 pieces using a cork borer (1 cm in diameter), then put into a culture bottle containing 500 mL of sterile PDB medium (Kumala and Pratiwi, 2014), incubated at room temperature for 4 weeks. After 4 weeks, each cultivated endophytic fungi biomass was determined the wet biomass, then dried using an oven at 60 oC to obtain the dried biomass. The media of each endophytic fungi isolates were mixed using 500 mL of ethyl acetate. The secondary metabolites layer (at the top) was concentrated using a rotary evaporator at 73 oC to obtain the extract of endophytic fungi (Desmara et al., 2017).
2.6 Testing of the Antibacterial Activity using Kirby- Bauer Method
The suspension of the bacteria (E.coli, S.aureus and S.dysentriae) which was equivalent to the Mc Farland 0.5 standard was taken as much as 0.2 mL and poured into a sterile petri dish. Sterile MHA medium was poured into a petri dish containing bacterial suspension and waited until solid. Secondary metabolite extracts made into concentrations of 4000 µg/mL using DMSO. Paper discs saturated with 10 µl extracts of secondary metaboites concentrations of 4000 µg / mL, then placed on the MHA medium surface (Jamal et al., 2008).
Positive control used saturated paper discs with 10 µL tetracycline antibiotic with a concentration of 4000 µg/mL, while the negative control used saturated sterile paper discs with 10 µL DMSO. Then incubated in incubator for 24 hours at 37 oC. The diameter of the inhibitory zone formed includes the diameter of paper discs measured using a caliper (Sulaiman et al., 2017).
2.7 MIC Determination of the Secondary Metabolite Extracts of Endophytic Fungi
The secondary metabolites extracts of the the endophytic fungi which have the largest inhibitory zone diameter for each bacteria were carried out by MIC testing. MIC testing was carried out to determine the smallest concentration of the secondary metabolite extracts which still formed the inhibition zone to each bacteria. The concentrations of extracts used were 4000, 2000, 1000, 500, 250 and 125, 62.5, 31,25, 15.625, and 7.8125 µg/mL (Salni et al., 2016). The diameter of the inhibitory zone formed includes the diameter of paper discs measured using a caliper (Sulaiman et al., 2017).
2.8 Thin Layer Chromatography (TLC)
The secondary metabolites extracts of endophytic fungi which were tested by MIC were carried out by thin layer chromatography (TLC) analyses to determine the group of chemical compounds contained in the extracts.
Secondary metabolite extracts were dissolved with ethyl acetate solvents, then taken using capillary pipes and
Asiandu et al, 2019 |Abstract 27
dripped onto the TLC plate. Elution is carried out by the mobile phase of n-hexane: ethyl acetate (6:4). The plate was sprayed by 2% H2SO4 and heated on a hot plate until the spot colors appears (Hidayati, 2012). The Rf value was calculated based on the formula (Sumarto et al., 2011):
2.9 Characterization of Endophytic Fungi Isolates
One isolate of endophytic fungi which has the largest inhibiton zone to each bacteria based on antibacterial activity test was characterized based on morphological characters.
2.10 Macroscopic Characterization of Endophytic Fungi
Endophytic fungi were inoculated into petri dishes containing sterile CDA, CYA, MEA and PDA media, incubated at room temperature for 72 hours, observed for up to 7 days. The macroscopic characters of endophytic fungi were observed in colony color, colony diameter, and color opposite the colonies (Widowati et al., 2016).
2.11 Microscopic Characterization of Endophytic Fungi
Glass objects dripped with lactic acid. The endophytic fungal mycelium was taken using sterile osseous needle and scratched on the object glass and covered with a cover glass. Microscopic characters include hyphae, spores or conidia and producing bodies of spores or conidia (Sanjaya et al., 2010).
2.12 Identification of Endophytic Fungi Isolates
Identification of endophytic fungi isolates was using:
Compendium of Soil Fungi Volume 1. 1980. K.H.
Domsch, W. Gams & T-H. Andersen. 1980. Academic Press. London.
Training Course for the Identification of Aspergillus, Penicillium and Talaromyces. Robert A. Samson.
Westerdijk Fungal Biodiversity Institute, Utretch, the Netherlands.
3. Results and discussion
Isolation and Purification of Endophytic Fungi of Dragon’s Scales Fern
Based on the research, 13 isolates of endophytic fungi were obtained from dragon’s scale fern fronds. The isolates obtained from trophophyll fronds were 5 isolates include DTP1, DTP2, DTP3, DTP4 and DTG1. The endophytic fungi isolates obtained from sporophyll fronds were 8 isolates which included DSP1, DSP2, DSP3, DSP4, DSP5, DSP6, DSG1 and DSG2.
The amount of endophytic fungi isolates from sporophyll fronds were more than trophophyll fronds.
Sporophyll fronds contain more carbon sources than trophophyll fronds. According to Watkins et al. (2016), photosynthesis in trophophyll fronds is carried out through a CO2 fixation process that produces glucose as an energy source. The results of photosynthesis will be circulated to all parts of the body of the fern including the sporophyll fronds. Sporophyll fronds will accumulate glucose which produced in photosynthesis by trophophyll fronds. Thus sporophyll fronds have higher carbon source than trophophyll fronds, its respiration rate 4 times higher than trophophyll fronds.
Antibacterial Activity Test of Secondary Metabolite Extracts of Endophytic Fungi
The result of antibacterial activity test of secondary metabolites extracts of endophytic fungi are presented in Figure 1. Based on Figure 1. Secondary metabolites extracts of DTP2 isolate had the largest inhibitory zone diameter againts E.coli compared to other isolates which was 14,82 ± 4,05 mm. The diameter of the inhibitory zone formed on the medium was due to the diffusion of active compounds from secondary metabolites extract on paper discs to the medium. According to Erlyn (2016), the inhibition of the growth of bacteria is characterized by the presence of inhibition zones around the paper discs which are not overgrown by the bacteria, because of the paper discs containing antibacterial compounds.
The secondary metabolites extract of DSP4 isolate had the highest inhibition zone diameter against S.dysentriae which was 10,15 ± 0,36 mm. The diameter of the inhibitory zone of an extract against the bacteria can be influenced by several factors. Factors that affect the diameter size of the inhibition zone are the concentration of extract, the content of secondary metabolites extract and the type of bacteria. According to Lestari et al.
(2016), an extract of secondary metabolites containing antibacterial compounds will form an inhibition zone to the bacteria. The diameter of the inhibitory zone depend on the concentration of the extract used. Bacterial resistance also affects the presence or absence of the inhibitory zone.
Asiandu et al, 2019 |Abstract 28
Figure 1. Antibacterial activity of the secondary metabolite extracts of endophityc fungi from dragon’s scale fern fronds against E.coli, S.aureus and S.dysentriae
The inhibitory zone diameter of tetracycline against S.aureus based on Figure 1. was 8,50 ± 0,06 mm, was lower than the inhibitory zone of secondary metabolites extract of isolate of DTP4 with 8,80 ± 0,03 mm. This might be caused by the resistance of S.aureus to tetracycline. According to Chudlori et al. (2012), S.aureus are pathogenic bacteria that cause infectious diseases which have the ability to develop rapid resistance to various types of commonly used antibiotics. S.aureus are able to transfer antibiotic resistance plasmid through an intrinsic resistance mechanism.
MIC Determination of Secondary Metabolite Extracts of Endophytic Fungi
Based on MIC results, the smallest concentration of secondary metabolites extracts of isolate of DTP2 which still formed the inhibitory zone to E.coli was 125 µg/mL with diameter of inhibitory zone was 6.10 ± 0.00 mm.
Based on the results of the MIC determination value, antibacterial activity of the extract of DTP2 against E.coli was classified as strong. MIC determination result of the extract of secondary metabolites of DSP4 against Shigella dysentriae was 31.25 µg/mL with diameter 6.20 ± 0.01 mm, the antibacterial activity was classified as very strong. MIC value of the secondary metabolites extract of DSP4 against S.aureus was 125 µg/mL with inhibitory zone diameter 6.90 ± 0.00 mm, and the antibacterial activity was classified as strong.
The antibacterial activity of an extract based on the MIC determination value classified into five categories includes very strong, strong, medium, weak and inactive.
According to Saraiva et al. (2011), antibacterial compound which has MIC value less than 100 µg/mL classified as very strong antibacterial. Antibacterial compound which has MIC value 100-500 µg/mL classified as strong. MIC value of an antibacterial between 1000-2000 µg/mL classified as weak and the MIC value of an extract more than 2000 µg/mL classified as inactive compound.
The decrease of an extract concentration will affect the inhibitory zone diameter formed. The lower the concentration of the secondary metabolites extract used, the smaller inhibitory zone formed. Based on Lingga et al.
(2015), the decrease of an extract concentration influences the inhibitory zone diameter. The lower the concentration of the secondary metabolites extract used, the smaller inhibitory zone formed, because of the amount of active compounds of an extract depends on the concentration of the extract.
Thin Layer Chromatography (TLC) Analyses
The results of TLC analyses of this research are presented on Figure 2. Based on Figure 2. Secondary metabolites extract of endophytic fungi of isolate DTP2 contains chemical compounds includes tannins, terpenoids and phenolics. Secondary metabolites extract of endophytic fungi of isolate DTP4 identified contains chemical compounds includes tannins, terpenoids, steroids and phenolics. Secondary metabolites extract of endophytic fungi of isolate DSP4 contains alkaloids, terpenoids, phenolics and tannins.
Chemical compounds of a secondary metabolite extract can be detected by the spots color which are appeared on TLC plates. Each spot color indicate the secondary metabolite compounds of the extract.
According to Normansyah et al. (2013), terpenoids detected by the purple spot color after sprayed by H2SO4
and heated. The alkaloids compounds are purplish or brick red, tannins have brownish spots color and steroids are green. Yellow spots on the TLC plate prove the presence of phenolics compounds.
Alkaloids are able to damage the bacterial cell components. According to Erlyn (2016), alkaloids play role as an antibacterial agent by damaging the constituent components of peptidoglycan on bacterial cell walls.
Based on Afni et al. (2015), tannins, able to shrink cell walls or bacterial cells thereby changing the permeability
Asiandu et al, 2019 |Abstract 29
of bacterial cells. Based on Lestari et al. (2016), steroids will damage the bacterial cell plasma membranes.
Based on Awanis and Mutmainnah (2016), low concentration of phenolics compounds able to form phenol-proteins weak bond. According to Haryati et al.
(2015), terpenoids bind the transmembrane protein on the outer membrane of bacterial cells.
Characterization and Identification of Endophytic Fungi Isolates
Based on the results of the macroscopic morphological characterization, the endophytic fungal isolate DTP2 on the PDA has 2 cm, greenish black colony. Colonies of the isolate of DTP2 which incubated for 7 days in the MEA were black, on the CDA greenish gray with a colony diameter of 3.35 cm. The colonies of endophytic fungi isolate DTP2 in CYA were dark green, 2.26 cm in diameter.
Figure 3. Isolate DTP2 on CDA (7 days incubation at room temperature)
The microscopic morphological characteristics of endophytic fungal isolate DTP2 as shown on the Table 1.
has chlamydospore, septate hyphae with a blackish brown color. Based on these morphological characters, the isolate of DTP2 are similiar to Aureobasidium melanogenum. The similiar characters of DTP2 was compared to the characters of A.melanogenum presented by Castiglia and Kuhar (2015). According to Castiglia and Kuhar (2015), A.melanogenum has blackish brown
hyphae, thin and insulated walls, ellipses conidia which has varying sizes. Vegetative hyphae was blackish brown, then develop to form dark brown, thick-walled chlamydospore, consisting of one or two cells.
Table 1. Microscopic characters of isolate DTP2
Based on the results of the macroscopic morphological characterization, the endophytic fungal isolate DTP4 after 7 days incubation on the PDA has white colonies, 2,1 cm in diameter and colonies reverse were yellow. The colonies of DTP4 on MEA were grayish green with a colony diameter 2.6 cm and colonies reverse were pale yellow. The colonies color of DTP4 on CDA were greenish gray, the colonies diameter were 1.34 cm, the medium color around the colonies was white and the reverse medium was pale yellow. While on the CYA, colonies of DTP4 have a dark green color, 2.56 cm in diameter, the medium color around the colonies was pale yellow, on the colonies reverse were yellow.
Figure 4. Isolate DTP4 on MEA (7 days incubation at room temperature)
The microscopic morphological characteristics of the isolate of DTP4 as shown on Table 2. were septate hyphae, terverticillate branched conidiophores, conidia
Asiandu et al, 2019 |Abstract 30
globose and shaped flask phialide. The microscopic morphological characters of DTP4 similiar to Penicillium alli-sativi as described by Houbraken et al. (2012).
According to Houbraken et al. (2012), P.alli-sativi has terverticillate or quarterverticillate branched conidiophores. Smooth walls stipe, each metula has the same length, ampulliform phialide, and soft textured globose or subglobose conidia.
Table 2. Microscopic characters of isolate DTP4
Morphological characters of endophytic fungi isolate DSP4 on PDA after 7 days incubation was white colonies, 2.7 cm in diameter. The colonies of DSP4 on MEA were yellowish white, 2.6 cm in diameter. The colonies were white on CDA, 1,4 cm. While on the CYA the colonies were white orange, 3.62 cm.
Figure 5. Isolate DSP4 on CYA (7 days incubation at room temperature)
The microscopic morphological characters of DSP4 as shown on the Table 3 were radiate conidial head, globose conidia, septate hypae, and brownish sclerotia.
The microscopic morphological characters of DSP4 similiar to Aspergillus flocculosus as described by Frisvad et al. (2004). According to Frisvad et al. (2004),
A.flocculosus has radiate conidial head, globose vesicles covered by metulae, globose, smooth, and tiny conidia.
Globose to subglobose sclerotia, covered by mycelium.
Table 3. Microscopic morphological characters of isolate DSP4
Microscopic morphological characters of DSP4 as shown on Table 3. were radiate conidial head, globose conidia, septate hypae and brownish sclerotia. The characters have the similiar characters to characteristics
Antibacterial activity of A.melanogenum was reported by Sepcic et al. (2011), the secondary metabolites extract of A.melanogenum was able to inhibit the growth of Bacillus subtilis. According to Houbraken et al. (2012), P.alli-sativi are able to produce secondary metabolites which are potentially as antibacterial agents such penicillins. Based on Trinh et al. (2017), A.flocculosus has antibacterial activity by producing some antibacterial secondary metabolites including ochratoxin A and B, penicillic acid, asteltoxin, xanthomegnin, viomellin and vioxhantin.
4. Conclusion
1. The isolated endophytic fungi isolate from dragon’s scales fern fronds were 13 isolates consisting of 5 isolates from trophophyll fronds and 8 isolates from sporophyll fronds.
2. The secondary metabolites extract of endophytic fungi from dragon’s scales fern which potentially as an antibacterial agent were DTP2 against E.coli, DTP4 against S.aureus and DSP4 against S.dysentriae.
3. MIC value of secondary metabolites ethyl acetate extracts of endophytic fungi isolate DTP2 against E.coli was 125 μg/mL, DTP4 against S.aureus was 125
Asiandu et al, 2019 |Abstract 31
μg/mL and DSP4 against S.dysentriae was 31,25 μg/mL.
4. Endophytic fungi isolate DTP2 identified as Aureobasidium melanogenum, DTP4 identified as Penicillium allii-sativi and DSP4 identified as Aspergillus flocculosus.
References
[1] Afni, N., N. Said and Yuliet. 2015. Uji Aktivitas Antibakteri Pasta Gigi Ekstrak Biji Pinang (Areca catechu L.) terhadap Streptococcus mutans dan Staphylococcus aureus. Journal of Pharmacy.
1(1): 48-58.
[2] Akmalasari, I., E. S. Purwati and R. S. Dewi.
2013. Isolasi dan Identifikasi Jamur Endofit Tanaman Manggis (Garcinia mangostana L.).
Jurnal Biosfera. 30(2): 82-89.
[3] Awanis, M. A. and A. A. Mutmainnah. 2016. Uji Antibakteri Ekstrak Oleoresin Jahe Merah (Zingiber officinale var. rubrum) terhadap Bakteri Steptococcus pyogenes. 3(1): 33-41.
[4] Castiglia, V. C. and F. Kuhar. 2015. Deterioration of Expanded Polystyrene Caused by Aureobasidium pululans var. melanogenum.
Jurnal Revista Argentia de Microbiologia. 47(3):
256-260.
[5] Chudlori, B., M. Kuswandi and P. Indrayudha.
2012. Pola Kuman dan Resistensinya Terhadap Antibiotika dari Spesimen Pus di RSUD Dr.
Moewardi Tahun 2012. Jurnal Pharmacon. 13(2):
70-76.
[6] Deepika, V. B., T. S. Murali and K.
Satyamoorthy. 2016. Modulation of Genetic Clusters for Synthesis of Bioactive Molecules in Fungal Endophytes: a Review. Jurnal Michrobiological Research. 182(1): 125-140.
[7] Desiyana, L. S., M. A. Husni and S. Zhafira.
2016. Uji Efektivitas Sediaan Gel Fraksi Etil Asetat Daun Jambu Biji (Psidium guajava Linn.) terhadap Penyembuhan Luka Terbuka pada Mencit Mus musculus). Jurnal Natural. 16(2): 23- 32.
[8] Desmara, S., S. Rezeki and Sunnati. 2017.
Konsentrasi Hambat Minimum dan Konsentrasi Bunuh Minimum Ekstrak Daun Kemangi (Ocimum sanctum L.) terhadap Pertumbuhan Candida albicans. Jurnal Caninus Denstistry.
2(1): 31-39.
[9] Erlyn, P. 2016. Efektivitas Antibakteri Fraksi Aktif Serai (Cymbopogon citratus) terhadap Bakteri Streptococcus mutans. Jurnal Syifa’
Medika. 6(2): 111-125.
[10] Erviani, A. E. 2013. Analisis Multidrug Resistensi terhadap Antibiotik pada Salmonella
typhi dengan Teknik Multiplex PCR. Jurnal Biogenesis. 1(1): 51-60.
[11] Frisvad, J. C., J. M. Frank., J. A. M. P.
Houbraken., A. F. A. Kuijpers and R. A. Samson.
2004. New Ochratoxin a Producing Species of Aspergillus Section Circumdati. Jurnal Studies in Mycology. 50: 23-43.
[12] Haryati, N. A., C. Saleh and Erwin. 2015. Uji Toksisitas dan Aktivitas Antibakteri Ekstrak Daun Merah Tanaman Pucuk Merah (Syzygium myrtifolium Walp.) terhadap Bakteri Staphylococcus aureus dan Escherichia coli.
Jurnal Kimia Mulawarman. 13(1): 35-40.
[13] Hidayati, N. 2012. Isolasi dan Penetapan Kadar Senyawa Antifungal p-Methoxybenzylidene p- aminophenol dari Akar Acacia mangium. Jurnal Pemuliaan Tanaman Hutan. 6(2): 117-130.
[14] Houbraken, J., J. C. Frisvad., K. A. Seifert., D.
P. Opery., D. M. Tuthill., J. G. Valdez and R. A.
Samson. 2012. New Penicillin-producing Penicillium Spesies and an Overview of Section Chrysogena. Jurnal Persoonia. 29: 78-100.
[15] Jamal, Y., M. Ilyas., A. Kanti and A. Agusta.
2008. Diversitas dan Profil Metabolit Sekunder Jamur Endofit yang Diisolasi dari Tumbuhan Gambir (Uncaria gamble) serta Aktivitas Biologisnya sebagai Antibakteri. Jurnal Ilmiah Nasional Berita Biologi. 9(1): 149-154.
[16] Kumala, S. and A. A. Pratiwi. 2014. Efek Antimikroba dari Kapang Endofit Ranting Tanaman Biduri. Jurnal Farmasi Indonesia. 7(2):
111-120.
[17] Lestari, Y., P. Ardiningsihand Nurlina. 2016.
Aktivitas Antibakteri Gram Positif dan Negatif dari Ekstrak dan Fraksi Daun Nipah (Nypa fruticans Wurmb.) Asal Pesisir Sungai Kakap Kalimanta Barat. Jurnal Kimia Khatulistiwa.
5(4):1-8.
[18] Lingga, A. R., U. Pato and E. Rossi. 2015. Uji Antibakteri Ekstrak Batang Kecombrang (Nicolaia speciosa Horan) terhadap Staphylococcus aureus dan Escherichia coli.
Jurnal JOM Faperta. 3(1): 1-15.
[19] Murdiyah, S. 2017. Fungi Endofit pada Berbagai Tanaman Berkhasiat Obat di Kawasan Hutan Evergreen Taman Nasional Baluran dan Potensi Pengembangan sebagai Petunjuk Praktikum Mata Kuliah Mikologi. Jurnal Pendidikan Biologi Indonesia. 3(1): 64-71.
[20] Normansyah, A., N. P. Ariantari and K. W.
Astuti. 2013. Profil Kandungan Kimia Ekstrak Etanol 80% Kulit Batang Michelia champaca L.
dengan Kromatografi Lapis Tipis dan Pereaksi Pendeteksi. Jurnal Farmasi Udayana. 2(3): 153- 156.
Asiandu et al, 2019 |Abstract 32
[21] Nugrahani, R., Y. Andayani and A. Hakim.
2016. Skrining Fitokimia dari Ekstrak Buah Buncis (Phaseolus vulgaris L.) dalam Sediaan Serbuk. Jurnal Penelitian Pendidikan IPA. 2(1):
96-103.
[22] Oktavia, S., H. Arifin and E. Duarto. 2017.
Pengaruh Pemberian Ekstrak Etanol Daun Sisik Naga (Pyrrosia pilloselloides (L.) M. G. Price) terhadap Waktu Pendarahan, Waktu Pembekuan Darah dan Jumlah Trombosit Mencit Putih Jantan.
Jurnal Farmasi Higea. 9(1): 48-55.
[23] Pratiwi, E., U. Hasanah dan Indramsyah. 2014.
Identifikasi Senyawa metabolit Sekunder pada Jamur Endofit dari Tumbuhan Raru (Cotylelobium melanoxylon). Prosiding Seminar Nasional Biologi dan Pembelajarannya Universitas Negeri Medan. 267-277.
[24] Purwanto, S. 2015. Uji Aktivitas Antibakteri Fraksi Aktif Ekstrak Daun Senggani (Melastoma malabathricum L.) terhadap Escherichia coli.
Jurnal Keperawatan Sriwijaya. 2(2): 84-92.
[25] Ramadhani, S. H., Samingan and Iswadi. 2017.
Isolasi dan Identifikasi Jamur Endofit pada Daun Jamblang (Syzygium cumini L.). Jurnal Ilmiah Mahasiswa FKIP Unsyiah. 2(2): 77-90.
[26] Salni., H. Marisa and Harmida. 2016. Uji Aktivitas Bahan Bioaktif dari Daun Salung (Psychotria viridiflora Reinw Ex. Blume) terhadap Bakteri Salmonella tyhpi secara in Vitro dan In Vivo. Jurnal Ilmu Kefarmasian Indonesia.
14(1): 13-18.
[27] Sanjaya, Y., H. Nurhaeni and M. Halma. 2010.
Isolasi, Identifikasi dan Karakterisasi Jamur Entomopatogen dari Larva Spodoptera litura (Fabricius). Bionaruta- Jurnal Ilmu-ilmu Hayati dan Fisik. 1293): 136-141.
[28] Saptarini, N. M., D. Suryasaputra and N.
Nurumamah. 2018. Antioxidant Activity of Extract and Fractions of Dragon Scales (Drymoglossum piloselloides C. Presl.). Research Journal of Pharmaceutical, Biological, and Chemical Science. 9(1): 757-763.
[29] Saraiva, A. M., R. H. A. Castro., R. P. Cordeiro., T. J. S. P. Sobrinho., V. T. N. A. Castro., E. L. C.
Amorim., H. S. Xavier and M. N. C. Pisciottano.
2011. In Vitro Antioxidant, Antimicrobial and Toxicity Properties of Schinopsis brasiliensis Engl. (Anacardiaceae). African Journal of Pharmacy and Pharmacology. 5(14): 1724-1731.
[30] Sepcic, K., P. Zalar and N. Gunde-Cimerman.
2011. Low Water Activity Includes the Production of Bioactive Metabolites in Halophilic and Halotolerant Fungi. Marine Drugs. 9: 43-57.
[31] Suciatmih., Yuliar and D. Supriyati. 2011.
Isolasi, Identifikasi dan Skrining Jamur Endofit Penghasil Agen Biokontrol dari Tanaman di
Lahan Pertanian dan Hutan Penunjang Gunung Salak. Jurnal Tek Ling. 12(2): 171-186.
[32] Sulaiman, A. Y., P. Astuti and A. D. P. Shita.
2017. Uji Antibakteri Ekstrak Daun Kersen (Muntingia calabura L.) terhadap Koloni Streptococcus viridans. Indonesian Journal for Health Sciences. 1(2): 1-6.
[33] Watkins, J. E., A. C. Churchill and N. M.
Holbrook. 2016. A Site for Sori: Ecophysiology of Fertile-Sterile Leaf Dimorphy in Ferns.
American Journal of Botany. 103(5): 1-11.
[34] Widowati, T., Bustanussalam., H. Sukiman and P. Simanjuntak. 2016. Isolasi dan Identifikasi Kapang Endofit dari Tanaman Kunyit (Curcuma longa L.) sebagai Penghasil Antioksidan. Jurnal BIOPROPAL Industri.7(1): 9-16.